1. Field of the Invention
The present invention relates generally to improved therapeutic metal chelating agents which are highly effective and have low toxicity upon injected and oral administration, and in particular to chelating agents which incorporate within their structures 3-hydroxy-2-pyridinone (3,2-HOPO) moieties with a carbamoyl group substituted on the ring carbon atom ortho to the hydroxy or oxo group of the HOPO ring.
2. Description of Related Art Including Information Disclosed Under .sctn..sctn. 1.97-1.99
Siderophores are highly selective and effective ferric chelating agents synthesized and released by microorganisms to ensure the presence of sufficient iron in solubilized form for cell reproduction. It was recognized early on that the affinity and selectivity of the siderophores for ferric ion made these compounds good candidates for therapeutic iron removal agents. This is particularly true for patients who suffer from blood diseases such as beta thalassemia, the treatment of which requires the regular transfusion of whole blood and results in the accumulation of massive tissue iron deposits. Because of the similarity in coordination properties between Fe(III) and tetravalent actinides, tetravalent actinides have great affinity for electron-donor groups that bind Fe(III), and follow Fe(III) in mammalian iron transport and storage systems. The great affinity and specificity of the siderophores towards Fe(III) suggest that modification of siderophores, which are effective sequestering agents for ferric ion, would yield potential chelators of tetravalent actinides, which present significant biological hazards associated with nuclear technology. Following absorption, the actinide cations that have been inhaled, ingested, or deposited in a wound circulate in serum bound to transferrin (Tf), the iron transport protein, and renal and gastrointestinal excretion are severely inhibited. As actinide-containing cells and structures die, the released actinide is recirculated, and nearly all of it is re-deposited at new sites. The alpha particles emitted by the actinides kill cells and induce cancer in the major storage tissues--lung, bone, liver. The only known way to reduce the toxicity of these radioactive metals is to use chelating agents to accelerate their excretion, thereby preventing deposition or re-deposition. Normally, such actinide chelating agents will be octadentate ligands, as opposed to the generally hexadentate or tetradentate siderophores. Other uses, such as radionuclide chelation in nuclear medicine applications, for example, are also clearly possible.
The biomimetic approach of the present invention, which designs and synthesizes sequestering agents for ferric ion and actinides, is based on siderophores. The metal binding units of siderophores are usually either catechols (dihydroxybenzene analogs; Formula 1A) or hydroxamic acids (Formula 1B): ##STR1## In fact, desferrioxamine B (DFO), a tri-hydroxamic acid siderophore, is used as a human iron sequestering agent. This chelating agent has predominated for over 30 years as the method of choice for treatment of iron overload. However, DFO has low oral activity and a number of adverse effects: including administration via a cumbersome subcutaneous infusion, leading to poor patient compliance with the treatment regime, and poor efficacy in removing deposited actinides. As a result of these limitations of the prior art drugs, there is a need for more effective and orally active iron sequestering agents to treat iron overload as well as actinide poisoning.
The most potent natural Fe(III) chelator is enterobactin, a siderophore produced by enteric bacteria with a formation constant of K.sub.f .apprxeq.10.sup.49, pM=35.5. This hexadentate ligand is composed of three catechoylamide groups attached to a tri-serine lactone backbone. Catecholates are much stronger sequestering agents than hydroxamate ligands, such as DFO, and these ligands are faster in removing iron from human transferrin, primarily for kinetic rather than thermodynamic reasons. Synthetic analogues of catechol-based siderophores are also known. However, there are a number of difficulties in developing catecholates into effective pharmaceutical agents. A number of catecholate siderophores, including enterobactin, will be bound by albumin in serum. They also strongly promote the growth of pathogenic microorganisms. The weak acidity of catechol and the required loss of two protons per catechol group at or about neutral pH limit the effectiveness of catechol-based ligands in vivo. These factors place severe limitations on the use of catechol-based ligands as therapeutic agents. It is therefore desirable to provide a medicinally useful metal chelating agent having a higher K.sub.a, i.e., more acidic, and which therefore binds more effectively at physiological pH, than catechol-based compounds. Uninegative ligands, i.e., ligands having a single negative charge near neutral pH range, are particularly desirable, in contrast to the correspondingly highly charged ferric and plutonium catechol complexes.
Derivatives of hydroxypyridinones ("HOPO") are of particular interest, since these ligands selectively display high affinity for ferric and actinide ion. These ligands and their mono-anions have a zwitterionic resonance form that is isoelectronic with the catechol dianion. The abbreviation "HOPO" will hereinafter be used to include hydroxypyridinone analogs as well as isomers or tautomers thereof, in either protonated or deprotonated forms.
The HOPO ligands have been shown to be very promising sequestering agents. The bidentate 3,4-HOPO ligand, 1,2-dimethyl-3-hydroxy-4-pyridinone, is orally active and has gone through extensive study, including clinical trials. However, there are many limitations for such a simple bidentate ligand. Multidentate HOPO derivatives have advantages over simpler bidentate ligands: in particular, low toxicity resulting from a higher binding affinity (pM) at low (clinical level) ligand concentrations.
Previous patents on hydroxypyridone ligands used as chelating agents include "Hydroxypyridonate Chelating Agents", U.S. Pat. No. 4,698,431, patented by Kenneth N. Raymond, Robert C. Scarrow, and David L. White, Oct. 6, 1987. This invention provided 1,2-HOPO derivatives with either an amide or a carboxylic acid moiety in the number 6 position. These chelating agents are useful in selectively removing certain cations from solution and are particularly useful as ferric ion and actinide chelators. However, U.S. Pat. No. 4,698,431 did not claim other chelating agents having 3,2-HOPO moieties incorporated within their structures or a carboxy moiety on the number 3 position of 1,2-HOPO ring.
Other related art includes Pharmaceutical Compositions of Hydroxypyridones, U.S. Pat. No. 4,666,927, patented by Robert C. Hider, George Kontoghiorghes, Jack Silver, and Michael A. Stockham, May 19, 1987. Claim 1 of this patent claims a number of possible chelating agents having 1,2-HOPO, 3,2-HOPO, or 3,4-HOPO moieties incorporated within their structures that are linked through a number of possible combinations of linking groups, including --CONH-- groups. However, U.S. Pat. No. 4,666,927 teaches against a HOPO moiety having a substitution ortho to the hydroxy or oxo group of the HOPO ring.
In contrast to U.S. Pat. No. 4,666,927, the inventors have developed a new design strategy, that is to synthesize a new series of 3,2-HOPO derivatives with either a carboxylic acid or a (substituted) carbamoyl moiety substituted on the ring carbon ortho to the HOPO hydroxy group. The particular coordination geometry and the hydrogen bonding between the amide proton and HOPO oxygen donor in these HOPO-metal complexes disclosed by the present invention thereby make the new series of 3,2-HOPO derivatives unusually good complexing agents having very high stability and specificity towards metal binding. The inventors further found these new compounds have stronger acidity and chelating ability for iron and actinides and have high oral activity in removing toxic actinides in vivo.
Furthermore, the method of synthesizing the present invention having 3,2-HOPO moieties incorporated within their structures with the (substituted) carbamoyl group ortho to hydroxy group of HOPO ring is not obvious. One earlier attempt by the inventors included: reacting 4-carboxy-3-hydroxy-2(1H)-pyridinones (Formula 9A) with 1, 1'-carbonyldiimidizole to produce the active amide intermediate, which is then combined with backbone amines to form the corresponding novel 3,2-HOPO ligands, similar to the case of thiohydroxamate. See, e.g., Kamal Abu-Dari and Kenneth N. Raymond, "Ferric Ion Sequestering Agents. 23. Synthesis of Tris(hydroxypyridinethione) Ligands and Their Ferric Complexes; X-ray Structure Analysis of N,N',N"-Tris(((1,2-didehydro-1-hydroxy-2-thioxopyrid-6-yl)carbonyl)-2,2',2 "-triaminotriethylaminato)iron(III)," Inorg. Chem. 1991, 30, 519-524. However, the purification of the final product is difficult, therefore, this method is not preferred. A second attempt to carry out the above reaction produced the acid chloride of 1-alkyl-4-carboxy-3-hydroxy-2(1H)-pyridinone as an active intermediate using thionyl chloride or oxalyl chloride, similar to the case of catechoylamide ligands. Due to the low yield of compound in preliminary tests, this method is also not preferred.
The present invention discloses a process to synthesize the desired multidentate 3,2-HOPO ligand in good yield.
Accordingly the present invention comprises an effective multidentate siderophore analog HOPO ligand in which one or more HOPO rings are linked to a molecular or a polymeric backbone through amide linkages. The inventors have previously reported the synthesis of siderophore analogs with linear, multipedal and macrocyclic topologies, and have shown a more effective ligand is one with a greater predisposition toward binding. In the design of the present invention, these synthetic strategies, as well as the binding abilities, solubility and lipophilicity of the resulted compounds, are important factors considered.